In the isolation of desoxyribonucleic acids (DNA) or ribonucleic acids (RNA) from biological tissues or cells, the nucleic acids must be separated from the remaining tissue or cell constitutents such as proteins, lipids, etc. To this end, a tissue or cell extract is prepared from which the nucleic acids can be separated in a two-phase system (Mainwaring et al., in "Nucleic Acid Biochemistry and Molecular Biology", Blackwell Scientific Publications Oxford (GB), pp. 75-76 [1982]). Two methods are most commonly used which can also be combined (Maniatis et al., in "Molecular Cloning--A Laboratory Manual", Cold Spring Harbor Laboratory, pp. 458-460 [1982]).
In one method, a water/phenol two-phase system is used in which proteins and peptides are precipitated or dissolved in the organic phase while nucleic acids remain in the aqueous phase, provided that a specific salt concentration and a neutral pH are maintained. The second method is the chloroform/isoamyl alcohol or chloroform/octanol method in which the nucleic acids also remain in the aqueous phase, while the other, denatured macromolecules are precipitated at the interface between the phases.
It is essential in the isolation of nucleic acids using these two-phase system methods that the aqueous and organic phases be thoroughly intermixed and then separated again. To achieve a rapid phase separation, centrifugation is usually carried out. In the isolation of intact chromosomal DNA care must also be taken to ensure that, upon mixing, the DNA is not cleaved into small fragments by excessive shearing forces.
After the phases are separated, the organic phase is removed and, where required, the aqueous phase is again extracted with an equal volume of phenol, chloroform/isoamyl alcohol or chloroform/octanol. The extraction of the aqueous phase with the organic phase is carried out until the nucleic acids can be precipitated from the aqueous phase in a pure form with alcohol (e.g., ethanol or isopropanol). Of course, the extraction of nucleic acids in this manner requires a large expenditure of time because of the repeated mixing and centrifugation cycles.